Polymer stabilized neuropeptides

ABSTRACT

A substantially hydrophilic conjugate is provided having a peptide that is capable of passing the blood-brain barrier covalently linked to a water-soluble nonpeptidic polymer such as polyethylene glycol. The conjugate exhibits improved solubility and in vivo stability and is capable of passing the blood-brain barrier of an animal.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is related to commonly owned copendingProvisional Applications Serial No. 60/157,503, filed Oct. 4, 1999, andSerial No. 60/166,589, filed Nov. 19, 1999, and claims the benefit oftheir earlier filing dates under 35 U.S.C. Section 119(e).

FIELD OF THE INVENTION

[0002] The invention relates to a conjugate between a peptide andpolyethylene glycol or a substantially substitutable polymer and amethod of use thereof.

BACKGROUND OF THE INVENTION

[0003] There has been significant progress in the discovery anddevelopment of potential neuropharmaceuticals (small molecules,peptides, proteins, and antisense) for treating pain and brain disorderssuch as Alzheimer's and Parkinson's diseases over the last decade.However, systemic delivery of many newly discovered neuropharmaceuticalshas been hampered by the lack of an effective system for deliveringthem. Intravenous injection is usually ineffective because of inadequatetransport across the barrier between the brain and the blood supply (the“blood-brain barrier” or “BBB”). The blood-brain barrier is a continuousphysical barrier that separates the central nervous system, i.e., thebrain tissue, from the general circulation of an animal. The barrier iscomprised of microvascular endothelial cells that are joined together bycomplex tight intracellular junctions. This barrier allows the selectiveexchange of molecules between the brain and the blood, and prevents manyhydrophilic drugs and peptides from entering into the brain. Many of thenew potent neuroactive pharmaceuticals do not cross the BBB because theyhave a molecular weight above 500 daltons and are hydrophilic. Compoundsthat are non-lipophilic and have a molecular weight greater than 500daltons generally do not cross the BBB.

[0004] Several strategies for delivering high molecular weight,non-lipophilic drugs to the brain have been developed includingintracerebroventricular infusion, transplantation of geneticallyengineered cells that secrete the neuroactive compound, and implantationof a polymer matrix containing the pharmaceutical. See Pardridge, W. M.,J. Controlled Rel., (1996) 39:281-286. However, all of these involveinvasive surgical procedures that can entail a variety of complications.

[0005] Four nonsurgical transport mechanisms have been identified forcrossing the BBB, including: (i) transmembrane diffusion, (ii)receptor-mediated transport, (iii) absorptive-mediated endocytosis, and(iv) carrier-mediated transport. See Brownless et al., J.Neurochemistry, (1993) 60(3):793-803. Vascular permeability can beincreased by opening the tight junctions with hyperosmotic saccharidesolutions and analogs of bradykinin. An inherent problem in this methodis that undesirable compounds in the general circulation may enter thebrain through the artificially enlarged openings in the blood-brainbarrier.

[0006] It has been discovered that capillary endothelial cells in theblood-brain barrier have a high level of receptors to transferrin,insulin, insulin-like growth factor I and II, low-density lipoproteinand atrial natriuretic factor. See Friden, P. M., J. Controlled Rel.,(1996) 46:117-128. U.S. Pat. No. 5,833,988 to Friden describes a methodfor delivering a neuropharmaceutical or diagnostic agent across theblood-brain barrier employing an antibody against the transferrinreceptor. A nerve growth factor or a neurotrophic factor is conjugatedto a transferrin receptor-specific antibody. The resulting conjugate isadministered to an animal and is capable crossing the blood-brainbarrier into the brain of the animal.

[0007] U.S. Pat. No. 4,902,505 to Pardridge et al. describes the use ofchimeric peptides for neuropeptide delivery through the blood-brainbarrier. A receptor-specific peptide is used to carry a neuroactivehydrophilic peptide through the BBB. The disclosed carrier proteins,which are capable of crossing the BBB by receptor-mediated transcytosis,include histone, insulin, transferrin, insulin-like growth factor I(IGF-I), insulin-like growth factor II (IGF-II), basic albumin, andprolactin. U.S. Pat. No. 5,442,043 to Fukuta et al. discloses using aninsulin fragment as a carrier in a chimeric peptide for transporting aneuropeptide across the blood-brain barrier.

[0008] Non-invasive approaches for delivering neuropharmaceutical agentsacross the BBB are typically less effective than the invasive methods inactually getting the agent into the brain. High doses of the chimericpeptides are required to achieve the desired therapeutic effect becausethey are prone to degradation. The concentration of the chimericpeptides in the blood circulation can be quickly reduced by proteolysis.An aqueous delivery system is not generally effective for deliveringhydrophobic drugs.

[0009] Another method for delivering hydrophilic compounds into thebrain by receptor-mediated transcytosis is described by Pardridge et al.in Pharm. Res. (1998) 15(4):576-582. A monoclonal antibody to thetransferrin receptor (OX26 MAb) modified with streptavidin is used totransport the cationic protein, brain-derived neurotrophic factor (BDNF)through the BBB. BDNF is first modified with PEG²⁰⁰⁰-biotin to formBDNF-PEG²⁰⁰⁰-biotin, which is then bound to the streptavidin-modifiedantibody OX26 MAb. The resulting conjugate was shown to be able to crossthe BBB into the brain.

[0010] Enhancing the duration of antinociceptive effects in animals mayresult in less frequently administered analgesics, which can improvepatient compliance and reduce potential side effects. Maeda et al. inChem. Pharm. Bull. (1993) 41(11): 2053-2054, Biol. Pharm Bull. (1994)17(6):823-825, and Chem. Pharm. Bull. (1994) 42(9):1859-1863 demonstratethat by attaching polyethylene glycol amine 4000 to the C-terminalleucine of Leu-enkephalin (distant from the tyrosine residue needed forantinociception), they could increase the potency and duration ofLeu-enkaphalin when it was directly administered to the brain byintracerebroventricular injection.

[0011] There is a need in the art to deliver neuroactive agents fromsystemic circulation across the blood-brain barrier and into the brainthat reduces or eliminates some of the drawbacks and disadvantagesassociated with the prior art.

SUMMARY OF THE INVENTION

[0012] This invention provides a method for delivering a peptide intothe brain of a human or other animal through the blood-brain barrier.The peptide to be delivered is bonded to a water soluble, non-peptidicpolymer to form a conjugate. The conjugate is then administered to ananimal into the blood circulation so that the conjugate passes acrossthe blood-brain barrier and into the brain. The water-solublenonpeptidic polymer can be selected from the group consisting ofpolyethylene glycol and copolymers of polyethylene glycol andpolypropylene glycol activated for conjugation by covalent attachment tothe peptide.

[0013] In one embodiment of this invention, a substantially hydrophilicconjugate is provided having a transportable analgesic peptide, i.e., ananalgesic peptide capable of passing the blood-brain barrier, covalentlylinked to a water-soluble, and nonpeptidic polymer such as polyethyleneglycol. The conjugate is capable of passing the blood-brain barrier ofan animal.

[0014] Suitable transportable peptides for use in this embodiment of theinvention can include dynorphins, enkephalins, endorphins, endomorphins,and biphalin. Typically, these small neuropeptides are susceptible todegradation inside the body in blood circulation and in the brain. Incontrast, when conjugated to polyethylene glycol or to a similarnonpeptidic, nonimmunogenic, water-soluble polymer having similarproperties, these peptides exhibit significantly increased stability.

[0015] In another embodiment of this invention, a composition isprovided comprising a conjugate of this invention as described above anda pharmaceutically acceptable carrier. The composition can be directlyadministered into the gneral circulation of an animal by any suitablemeans, e.g., parenteral injection, injection of intracerebral vein, andintranasal, pulmonary, ocular, and buccal administration.

[0016] In accordance with yet another embodiment of this invention, amethod is provided for delivering an analgesic peptide across theblood-brain barrier into the brain of an animal. The method comprisesproviding a conjugate of this invention as described above, andadministering the conjugate into the bloodstream of the host animal.

[0017] It has previously been considered that large hydrophilic polymerssuch as polyethylene glycol, when attached to a peptide that is capableof crossing the blood-brain barrier, would interfere with the transportof the peptide across the blood-brain barrier. In particular, it hasbeen believed that direct conjugation of large hydrophilic polymers to apeptide not only would increase the hydrophilicity but would also impairthe interaction between the peptide and its receptor or other structuresin the BBB by steric interference from the large polymer strands.

[0018] It has now been discovered that, although the conjugate issubstantially hydrophilic and contains a water-soluble and nonpeptidicpolymer, the conjugate is nevertheless capable of passing the bloodbrain barrier of an animal. As compared to its native state, peptidesconjugated to a water-soluble and non-peptidic polymer can exhibitreduced immunogenicity, enhanced water solubility, and increasedstability. In particular, peptides conjugated to polyethylene glycol inaccordance with this invention have a longer circulation time, reducedsusceptibility to metabolic degradation and clearance, and oncedelivered into the brain through the blood-brain barrier, exhibitextended lifetime in the brain. Thus, this invention allows effectivedelivery of analgesic peptides into human and other animal brains andcan significantly improve the efficacy of the peptides being delivered.

DETAILED DESCRIPTION OF THE INVENTION

[0019] As used herein, “passing the blood-brain barrier” or “crossingthe blood-brain barrier” means that, once administered into the bloodcirculation of an animal at a physiologically acceptable ordinarydosage, a conjugate or a peptide is capable of passing the blood-brainbarrier of the animal to such a degree that a sufficient amount of theconjugate or peptide is delivered into the brain of the animal to exerta therapeutic, antinociceptive, or prophylactic effect on the brain, orto affect the biological functioning of the brain to a detectabledegree. “Passing the blood-brain barrier” or “crossing the blood-brainbarrier” can also be used herein to mean that the conjugate or peptideis capable of being taken up by an animal brain to a degree that isdetectable by a suitable method known in the art, e.g., in situ brainperfusion as disclosed in Williams et al., J. Neurochem., 66 (3),pp1289-1299, 1996, which is incorporated herein by reference.

[0020] The conjugate of this invention normally is substantiallyhydrophilic. By the term “substantially hydrophilic,” it is intended tomean that the conjugate of-this invention does not contain asubstantially lipophilic moiety such as fatty acids or glycolipids.Fatty acids and glycolipids are used in the art to increase thelipophilicity of a molecule in order to increase the ability of themolecule to pass cell membranes.

[0021] The term “analgesic” as used herein means any chemical substancesthat are desirable for delivery into the brain of humans or otheranimals for purposes of alleviating, mitigating, or preventing pain inhumans or other animals, or otherwise enhancing physical or mental wellbeing of humans or animals. Analgesic peptides can be introduced intothe brain of an animal to exert a therapeutic, antinociceptive, orprophylactic effect on the biological functions of the animal brain, andcan be used to treat or prevent pain.

[0022] Agents not typically considered “analgesic” can be attached tothe peptide/polymer conjugate of the invention. For example, diagnosticor imaging agents can be attached to the conjugate. Fluoroscein,proteins, or other types of agents specifically targeted to a particulartype of cell or protein, such as monoclonal antibodies, can all be usedin the conjugate of this invention for diagnostic or imaging purposes.

[0023] As described below, when an agent is incapable of passing theblood-brain barrier, i.e., is non-transportable across the BBB, thentypically a peptide which is capable of passing the blood-brain barrier,i.e., is transportable across the BBB, will be used in a conjugate ofthis invention as a carrier.

[0024] In one embodiment of this invention, the peptide is atransportable analgesic peptide. As used herein, the term“transportable” means that the peptide is capable of crossing theblood-brain barrier of an animal as defined above. Thus, a conjugate isprovided comprising a transportable peptide bonded to a water-soluble,nonpeptidic, nonimmunogenic polymer, including polyethylene glycol.

[0025] The term “peptide” means any polymerized α-amino acid sequenceconsisting from 2 to about 40 amino acids having a peptide bond(—CO—NH—) between each amino acid that can impact the condition andbiological function of the brain of an animal. An analgesic peptidenormally is an endogenous peptide naturally occurring in an animal, orfragments or analogs thereof. However, non-endogenous peptides that canimpact the conditions and biological functions of animal brain are alsoincluded.

[0026] Many peptides are generally known in the art that are believed tobe capable of passing the blood-brain barrier. Examples of transportablepeptides that are believed to be capable of crossing the blood-brainbarrier after PEGylation in accordance with the invention include, butare not limited to, biphalin and opioid peptides such as dynorphins,enkephalins, endorphins, endomorphins etc. Many derivatives andanalogues of these transportable peptides can also be used in thepractice of the invention.

[0027] Opioid peptides are believed to be especially suitable forpractice of the invention. Opioid peptides exhibit a variety ofpharmacological activities, including among them pain relief andanalgesia.

[0028] Enkephalin is a pentapeptide having an amino acid sequence ofH-Tyr-Gly-Gly-Phe-Met-OH (methionine enkephalin) orH-Tyr-Gly-Gly-Phe-Leu-OH (leucine enkephalin). Many enkephalin analogshave been identified and synthesized which are specific to differenttypes of opiate receptors. See, e.g., Hruby and Gehrig, (1989) MedicinalResearch Reviews, 9(3):343-401. For example, U.S. Pat. No. 4,518,711discloses several enkephalin analogs including DPDPE, [D-Pen², D-Pen⁵]enkephalin, which is a cyclic enkephalin analog made by substituting thesecond and fifth amino acid residues of the natural pentapeptides witheither cysteine or with D- or L-penicillamine (beta,beta-dimethylcysteine) and joining the two positions by a disulfidebond. DPDPE has been shown to be able to pass the blood brain barrierinto the brain. See, e.g., Williams et al. (1996) Journal ofNeurochemistry, 66(3):1289-1299. U.S. Pat. No. 5,326,751 disclosesDPADPE prepared by substituting the glycine residue at the thirdposition of DPDPE with an alanine residue. Both of the patents areincorporated herein by reference.

[0029] Other enkephalin analogs include biphalin(H-Tyr-D-Ala-Gly-Phe-NH-)₂, which is a synthetic analog of enkephalinthat is a dimerized tetramer produced by coupling two units having theformula H-Tyr-D-Ala-Gly-Phe-OH at the C-terminus with hydrazine. Thedimeric form of enkephalin enhances affinity, and specificity to thedelta-opioid receptor. Dimeric enkephalin analogs are disclosed inRodbard et al. U.S. Pat. No. 4,468,383, the contents of which areincorporated herein by reference.

[0030] Dynorphins are another class of opioid peptides. Naturallyisolated dynorphin has 17 amino acids. Many dynorphin fragments andanalogs have been proposed in the art, including, e.g., dynorphin(1-10), dynorphin (1-13), dynorphin (1-13) amide, [D-Pro¹⁰] Dynorphin(1-11) (DPDYN), dynorphin amide analogs, etc. See, e.g., U.S. Pat. Nos.4,684,624, 4,62,941, and 5,017,689, which are incorporated herein byreference. Although such analgesic peptides are capable of transportingacross the blood-brain barrier, many of them have a very short half-lifedue to their susceptibility to biodegradation inside the body.

[0031] Even though polyethylene glycol normally has a large molecularweight and is hydrophilic, conjugation to the transportable peptides inthe absence of a lipophilic moiety does not interfere withtransportability of the peptides. The conjugated peptides remain capableof crossing the blood-brain barrier. Typically, upon administration intothe general circulation of an animal, the conjugate of the invention,comprising a transportable peptide bonded to polyethylene glycol or anequivalent polymer, is taken up by the brain at a much greaterpercentage as compared to an unconjugated form of the peptide. Thepeptides in the conjugates of this invention have increased stabilityand exhibit extended half-life inside the body.

[0032] In another embodiment of this invention, a conjugate is providedcomprising a first peptide, which is a transportable peptide, and asecond neuroactive agent linked to each other by polyethylene glycol oran equivalent polymer. This second neuroactive agent may or may not becapable of crossing the blood-brain barrier by itself. The transportablepeptide is used as a carrier to transport a non-transportableneuroactive agent across the blood-brain barrier into the brain of ananimal. The linking polymer serves not only as a linker but alsoincreases solubility and stability of the conjugate and reduces theimmunogenicity of both the neuropeptide and the other neuroactive agentto be delivered.

[0033] In accordance with the invention, the transportable peptide and,optionally, another neuroactive agent as described above, are covalentlylinked to a water-soluble and nonpeptidic polymer to form a conjugate ofthis invention. The water-soluble and nonpeptidic polymers suitable foruse in various aspects of this invention include polyethylene glycol,other polyalkylene glycols, and copolymers of polyethylene glycol andpolypropylene glycol.

[0034] As used herein, the term polyethylene glycol (“PEG”) is inclusiveand means any of a series of polymers having the general formula:

HO—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—OH

[0035] wherein n ranges from about 10 to 2,000. PEG also refers to thestructural unit:

—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—

[0036] wherein n ranges from about 10 to about 2000. Thus, by PEG ismeant modified PEGs including methoxy-PEGs; PEGs having at least oneterminal moiety other than a hydroxyl group which is reactive withanother moiety; branched PEGs; pendent PEGs; forked PEGs; and the like.

[0037] The polyethylene glycol useful in the practice of this inventionnormally has an average molecular weight of from about 200 to 100,000daltons. Molecular weights of from about 200 to 10,000 are somewhat morecommonly used. Molecular weights of from about 300 to 8,000, and inparticular, from about 500 to about 5,000 daltons, are somewhat typical.

[0038] PEG is useful in biological applications because it hasproperties that are highly desirable and is generally approved forbiological or biotechnical applications. PEG typically is clear,colorless, odorless, soluble in water, stable to heat, inert to manychemical agents, does not hydrolyze or deteriorate, and is generallynontoxic. Poly(ethylene glycol) is considered to be biocompatible, whichis to say that PEG is capable of coexistence with living tissues ororganisms without causing harm. More specifically, PEG, in itself, isnormally considered nonimmunogenic, which is to say that PEG does nottend to produce an immune response in the body. Desirable terminalactivating groups by which PEG can be attached to various peptidesshould not appreciably alter the nonimmunogenic character of the PEG, soas to avoid immunogenic effects. Desirable PEG conjugates tend not toproduce a substantial immune response or cause clotting or otherundesirable effects.

[0039] PEG is a highly hydrated random coil polymer that can shieldproteins or peptides from enzymatic digestion, immune system moleculesand cells, and can increase the hydrodynamic volume to slowreticuloendothelial system (RES) clearance. PEG is a useful polymerhaving the properties of water solubility as well as solubility in manyorganic solvents. The unique solubility properties of PEG allowconjugation (PEGylation) to certain compounds with low aqueoussolubility, with the resulting conjugate being water-soluble. However,PEGylation, which is conjugating a PEG molecule to another molecule, isnot without its difficulties. The effects of a particular PEG derivativeare not necessarily predictable. The result depends on the specificinteraction between a particular compound and the functionalnon-peptidic PEG polymer.

[0040] The polymer used in this invention normally can be linear orbranched. Branched polymer backbones are generally known in the art.Typically, a branched polymer has a central core moiety and a pluralityof linear polymer chains linked to the central core. PEG is commonlyused in branched forms that can be prepared by addition of ethyleneoxide to various polyols, such as glycerol, pentaerythritol andsorbitol. For example, the four-arm, branched PEG prepared frompentaerythritol is shown below:

C(CH₂—OH)₄ +nC₂H₄O→C[CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—OH]₄

[0041] The central moiety can also be derived from several amino acids.An example is lysine.

[0042] The branched polyethylene glycols can be represented in generalform as R(-PEG-OH)_(n) in which R represents the core moiety, such asglycerol or pentaerythritol, and n represents the number of arms.Suitable branched PEGs can be prepared in accordance with U.S. Pat. No.5,932,462, the contents of which are incorporated herein in theirentirety by reference. These branched PEGs can then be used inaccordance with the teachings herein.

[0043] Forked PEGs and related polymers should be useful in the practiceof the invention. The term “forked” is used to describe those PEGs thatare branched adjacent at least one terminus thereof. The polymer has abranched moiety at one end of the polymer chain and two free reactivegroups, one on each end of the branched moiety, for covalent attachmentto another molecule. Each reactive moiety can have a tethering group,including, for example, an alkyl chain, linking a reactive group to thebranched moiety. Thus, the branched terminus allows the polymer to reactwith two molecules to form conjugates. Forked PEGs and related forkedpolymers are described in copending, commonly owned U.S. patentapplication Ser. No. 09/265,989, which was filed Mar. 11, 1999 and isentitled Poly(ethylene glycol) Derivatives with Proximal ReactiveGroups. This pending patent application is incorporated by referenceherein in its entirety. The forked PEGs can be either linear or branchedin the backbone attached to the branched terminus.

[0044] Water-soluble, substantially nonimmunogenic, nonpeptidic polymersother than PEG should also be suitable for practice of the invention,although not necessarily with equivalent results. These other polymerscan be either in linear form or branched form, and include, but are notlimited to, other poly(alkylene oxides), including copolymers ofethylene glycol and propylene glycol, and the like. Exemplary polymersare listed in U.S. Pat. No. 5,990,237, the contents of which areincorporated herein by reference in their entirety. The polymers can behomopolymers or random or block copolymers and terpolymers based on themonomers of the above polymers, straight chain or branched.

[0045] Specific examples of suitable additional polymers include, butare not limited to, poly(acryloylmorpholine) (“PAcM”) andpoly(vinylpyrrolidone)(“PVP”), and poly(oxazoline). PVP andpoly(oxazoline) are well known polymers in the art and their preparationshould be readily apparent to the skilled artisan. PAcM and itssynthesis and use are described in U.S. Pat. Nos. 5,629,384 and5,631,322, the contents of which are incorporated herein by reference intheir entirety.

[0046] To couple PEG to a peptide, e.g., a transportable peptide, toform a conjugate of this invention, it is often necessary to “activate”the PEG to prepare a derivative of the PEG having a reactive group atthe terminus for reaction with certain moieties on the peptide. Manyactivated derivatives of PEG have been described in the art and can allbe used in this invention, although not necessarily with equivalentresults. An example of such an activated derivative is the succinimidylsuccinate “active ester”:

CH₃O-PEG-O₂C—CH₂CH₂—CO₂—NS

[0047] where NS=

[0048] The succinimidyl active ester is a useful compound because itreacts rapidly with amino groups on proteins and other molecules to forman amide linkage (—CO—NH—). For example, U.S. Pat. No. 4,179,337 toDavis et al. describes coupling of this derivative to proteins(represented as PRO-NH₂):

mPEG-O₂CCH₂CH₂CO₂NS+PRO—NH₂ →mPEG-O₂C—CH₂CH₂—CONH—PRO

[0049] Other activated PEG molecules known in the art include PEGshaving a reactive cyanuric chloride moiety, succinimidyl carbonates ofPEG, phenylcarbonates of PEG, imidazolyl formate derivatives of PEG,PEG-carboxymethyl azide, PEG-imidoesters, PEG-vinyl sulfone, activeethyl sulfone derivatives of PEG, tresylates of PEG, PEG-phenylglyoxal,PEGs activated with an aldehyde group, PEG-maleimides, PEGs with aterminal amino moiety, and others. These polyethylene glycol derivativesand methods for conjugating such derivatives to an agent are generallyknown in the art and are described in Zalipsky et al., Use ofFunctionalized Poly(Ethylene Glycol)s for Modification of Polypeptides,in Use of Polyethylene Glycol Chemistry: Biotechnical and BiomedicalApplications, J. M. Harris, Ed., Plenum Press, New York (1992), and inZalipsky, Advanced Drug Reviews (1995)16:157-182, all of which areincorporated herein by reference.

[0050] Typically, conjugation of a water-soluble, nonimmunogenic polymerto a peptide in accordance with this invention results in the formationof a linkage between the polymer and the peptide. The term “linkage” isused herein to refer to groups or bonds normally formed as a result of achemical reaction.

[0051] Covalent linkages formed in the practice of this invention can behydrolytically stable. The linkage can be substantially stable in waterand does not react with water at a useful pH, under physiologicalconditions, for an extended period of time, preferably indefinitely.Alternatively, the covalent linkage can also be hydrolyticallydegradable under physiological conditions so that the neuroactive agentcan be released from the PEG in the body of an animal, preferably afterit is delivered into the brain of the animal.

[0052] The approach in which drugs to be delivered are released bydegradation of more complex agents under physiological conditions is apowerful component of drug delivery. See R. B. Greenwald, Exp. Opin.Ther. Patents, 7(6):601-609 (1997). For example, conjugates of theinvention can be formed by attaching PEG to transportable peptidesand/or neuroactive agents using linkages that are degradable underphysiological conditions. The half-life of a PEG-neuroactive agentconjugate in vivo depends upon the type of reactive group of the PEGmolecule that links the PEG to the neuroactive agent. Typically, esterlinkages, formed by reaction of PEG carboxylic acids or activated PEGcarboxylic acids with alcohol groups on neuroactive agents, hydrolyzeunder physiological conditions to release the neuroactive agent. See,e.g., S. Zalipsky, Advanced Drug Delivery Reviews, 16:157-182 (1995).For example, in PCT Publication No. WO 96/23794, it is disclosed thatpaclitaxel can be linked to PEG using ester linkages and the linkedpaclitaxel can be released in serum by hydrolysis. Antimalarial activityof dihydroartemisinin bonded to PEG through a hydrolyzable ester linkagehas also been demonstrated. See Bentley et al., Polymer Preprints,38(1):584 (1997). Other examples of suitable hydrolytically unstablelinkages include carboxylate esters, phosphate esters, disulfides,acetals, imines, orthoesters, peptides and oligonucleotides.

[0053] Typically, the degradation rate of the conjugate should becontrolled such that substantial degradation does not occur until theconjugate passes into the brain of an animal. Many peptides in theirnative state are subject to substantial degradation in blood circulationand in organs such as liver and kidney. The hydrolytically degradablelinkages can be formed such that the half-life of the conjugate islonger than the time required for the circulation of the conjugate inthe bloodstream to reach the blood-brain barrier. Some minor degree ofexperimentation may be required for determining the suitablehydrolytically unstable linkage between specific neuroactive agents andPEG derivatives, this being well within the capability of one skilled inthe art once apprised of the present disclosure.

[0054] The covalent linkage between a peptide and a polymer can beformed by reacting a polymer derivative such as an activated PEG with anactive moiety on the peptide. One or more PEG molecules can be linked toone peptide.

[0055] Conversely, multiple peptides, including transportable peptidesand/or other types of neuroactive agents, can be linked to one PEGmolecule. Typically, such a PEG molecule has multiple reactive moietiesfor reaction with the peptide and neuroactive agents. For this purpose,bifunctional PEGs, pendant PEGs, and dendritic PEGs can all be used.Reactive PEGs have also been synthesized in which several activefunctional groups are placed along the backbone of the polymer. Forexample, lysine-PEG conjugates have been prepared in the art in which anumber of activated groups are placed along the backbone of the polymer.Zalipsky et al., Bioconjugate Chemistry, (1993) 4:54-62.

[0056] In one embodiment of this invention, a conjugate having adumbbell structure is provided wherein a transportable peptide or othertransportable neuroactive agent capable of passing the blood-brainbarrier of an animal is covalently linked to one end of a polyethyleneglycol molecule, and another neuroactive agent to be delivered into thebrain of an animal is linked to the other end of the PEG molecule. Thisother neuroactive agent can be a transportable peptide, or any otherneuroactive agent. Typically, it is not transportable and cannot initself pass the blood-brain barrier. Therefore, the transportablepeptide or other agent at one end of the PEG molecule acts as a carrierfor delivering the non-transportable neuroactive agent into the brain.For this purpose, bifunctional PEGs, either homobifunctional orheterobifunctional PEGs, can be used. As used herein “bifunctional PEG”means a PEG derivative having two active moieties each being capable ofreacting with an active moiety in another molecule. The two activemoieties can be at two ends of a PEG chain, or proximate to each otherat a forked end of a PEG chain molecule, allowing for steric hindrance,if any. Suitable transportable peptides for use in this invention aredescribed above including, but not limited to, dynorphins, enkephalins,biphalin, endorphins, endomorphins, and derivatives and analoguesthereof.

[0057] The conjugate of this invention can be administered to an animalfor purposes of treating, mitigating, or alleviating pain. Examples ofanimal hosts include, but are not limited to, mammals such as humans,and domestic animals including cats, dogs, cows, horses, mice, and rats.

[0058] The conjugate of this invention can be administered in anysuitable manner to an animal. For example, the conjugate can beadministered parenterally by intravenous injection, intramuscularinjection, or subcutaneous injection. Alternatively, the conjugate ofthis invention can also be introduced into the body by intranasal andpulmonary inhalation or by oral and buccal administration. Preferably,intravenous injection is utilized such that substantially all of theconjugate in an injection dose is delivered into the bloodstream of theanimal, through which the conjugate circulates to the blood-brainbarrier of the animal.

[0059] The conjugate can be injected in the form of any suitable type offormulation. For example, an injectable composition can be prepared byany known methods in the art containing the conjugate of this inventionin a solvent such as water or solution, including saline, Ringer'ssolution. One or more pharmaceutically acceptable carriers that arecompatible with the other ingredients in the formulation may also beadded to the formulation. Excipients, including mannitol, sodiumalginate, and carboxymethyl cellulose, can also be included. Otherpharmaceutically acceptable components, including antiseptics such asphenylethylalcohol; stabilizers such as polyethylene glycol and albumin;isotonizing agents such as glycerol, sorbitol, and glucose; dissolutionaids; stabilizing buffers such as sodium citrate, sodium acetate andsodium phosphate; preservatives such as benzyl alcohol; thickeners suchas dextrose, and other commonly used additives can also be included inthe formulations. The injectable formulation can also be prepared in asolid form such as lyophilized form.

[0060] The PEGylated transportable peptides of the invention can beadministered in a variety of formulations, including, for example,intranasal, buccal, and oral administration. The dosage of the conjugateadministered to a human or other animal will vary depending on theanimal host, the types of transportable peptides and/or neuroactiveagents used, the means of administration, and the symptoms suffered bythe animal. However, the suitable dosage ranges in a specific situationshould be readily determinable by a skilled artisan without undueexperimentation.

[0061] The invention is further illustrated by the following examples,which are intended only for illustration purposes and should not beconsidered in anyway to limit the invention.

EXAMPLE 1 Modification and Purification PEG-Dynorphin A

[0062] Dynorphin A (1-11)(H-Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-NH₂) (1.47 mg) wasdissolved in 0.25 ml deionized water and 0.25 ml of 25 mM NaP, pH 5.8buffer in a 1.5 ml microcentrifuge tube. The reagent,NHS-PEG_(2K)-Fluoroscein (1.0 mg), was added to the peptide solution inapproximately 2-fold mole excess. After 30 minutes of reaction time, 0.1ml of 25 mM sodium phosphate buffer, pH 7.4 was added and the reactionwas allowed to proceed at room temperature for 3 hours.

[0063] Conjugation of NHS-PEG_(2K)-Fluoroscein was monitored bycapillary electrophoresis (CE) and mass spectrometry (MALDI).Purification of the PEG-Dynorphin A conjugate was performed on a HiTrapSP cation exchange column from Amersham/Pharmacia using a gradientelution from 5 mM sodium phosphate buffer, pH 4.0 to 50 mM sodiumphosphate, 1.5M NaCl buffer, pH 7.5 in 53 minutes. Fractions werecollected and the contents were analyzed by MALDI. These fractions werepooled and stored frozen prior to in vivo assay.

EXAMPLE 2 Modification and Purification PEG-Endomorphin II

[0064] Endomorphin II (H-Tyr-Pro-Phe-Phe-NH₂, 2.3 mg) was dissolved in1.15 ml of 5 mM sodium phosphate buffer, pH 8.0. Modification ofEndomorphin II was performed in 1.5 hours at room temperature by addingmPEG₂₀₀₀-SPA (38 mg) in a 5 mole excess. The reaction mixture wasanalyzed by mass spectrometry (MALDI) to determine the extent ofmodification. MALDI was used to verify that the reaction betweenmPEG₂₀₀₀-SPA and Endomorphin II went to completion. The sample wasdialyzed against water using a 2000 MWCO membrane and lyophilized priorto in vivo assay.

EXAMPLE 3 In situ Perfusion, Capillary Depletion, Brain Extraction andProtein Binding Studies of PEG-Dynorphin A and PEG-Endomorphin II

[0065] The protocol for the rat brain perfusion experiments was approvedby the Institutional Animal Care and Use Committee at the University ofArizona. The in situ perfusion, capillary depletion, brain extract andprotein binding studies were carried out as previously reported(Williams et al., J. Neurochem., 66 (3), pp1289-1299, 1996).PEG-dynorphin A (PdynA) had a very high in situ uptake R_(Br) value of0.343±1.84. In contrast in situ perfusion with I¹²⁵ Dynorphin, gave avery high R_(Br) of approximately 0.96. The entire radioactivity wasrecovered in the solvent front of the subsequent HPLC, showing thatlabeled dynorphin A (1-11) rapidly degrades, probably to I¹²⁵Tyr.

[0066] Capillary depletion studies of the PdynA were carried out, andrevealed that approximately 88% of the radioactivity associated with thecapillary fraction rather than the brain parenchyma.

[0067] In situ uptake of PEG-endomorphin II (Pend) gave an R_(Br) valueof 0.057±0.008, similar to those previously reported for peptides.Subsequent capillary depletion showed that of the radioactivity enteringthe brain, 32% was associated with the capillary fraction with 67% inthe brain parenchyma.

[0068] The protein binding of Pend was studied using the centrifreefilter system. It was found that 30% of 25,000 dpm Pend was bound to a1% BSA solution.

[0069] The major contribution is that PEGylation improved brain andblood enzymatic stability dramatically. Endomorphin and dynorphin arevery unstable in either brain or blood with half-lives on the order ofminutes. After PEGylation, those half-lives increased to hours forendomorphin II. In the case of endomorphin II, the half-life in bloodplasma was 3.2 minutes, and brain tissue was 13 minutes. AfterPEGylation, those half-lives increased to greater than two hours.

EXAMPLE 4 Conjugation of PEG-Doxorubicin to Endomorphin I

[0070] Endomorphin I (H-Tyr-Pro-Trp-Phe-NH₂, 3.0 mg, 4.9E-6 moles) wasdissolved in 1 ml of 50 mM sodium phosphate, pH 8.2 buffer containing150 mM NaCl and 50 mM DTT. A four fold molar excess of Traut's reagent(2.7 mg) was added and was allowed to react at room temperature for 2hours. The thiol-modified endomorphin was purified from DTT and Traut'sreagent using a Superdex 30 size exclusion column (Pharmacia). Themodified endomorphin fractions were collected and lyophilized.

[0071] Doxorubicin hydrochloride (3.0 mg, 5.2E-6 moles) was dissolved in1.0 ml of 50 mM sodium phosphate, pH 7.2 buffer containing 150 mM NaCl.The pH of the solution was titrated to 8.0 with 0.1N sodium hydroxide. Aten molar excess of heterobifunctional PEG (NHS-PEG_(2K)-OPSS) was addedto the doxorubicin solution. The reaction was allowed to proceed at roomtemperature for 2 hours. OPSS-PEG_(2K)-doxorubicin was purified fromunreacted PEG and free doxorubicin using a Superdex 30 size exclusioncolumn. The OPSS-PEG_(2K)-doxorubicin fractions were collected andlyophilized.

[0072] The lyophilized powders of modified endomorphin I andOPSS-PEG_(2K)-doxorubicin were reconstituted in 50 mM sodium phosphatebuffer, pH 6.0. An equimolar amount of each solution was mixed togetherand the two were reacted at room temperature for 6 hours. Thedoxorubicin-PEG_(2K)-endomorphin conjugate was purified on a Superdex 30size exclusion column.

EXAMPLE 5 Conjugation of PEG to DPDPE

[0073] 3.0 mg of DPDPE (Tyr-D-Pen-Gly-Phe-D-Pen) was dissolved in 5 mlof anhydrous acetonitrile. A 20% molar excess of PEG reagent (eithermPEG-SPA 5K [27.9 mg] or mPEG-SPA 2K [11.1 mg]) and triethylamine (0.8μl) was added to the DPDPE. The reaction was allowed to proceed at roomtemperature under an argon atmosphere for 2 days. The sample was dilutedto 15 ml with deionized water and lyophilized. The PEG-DPDPE powder wasreconstituted in 5 ml of deionized water and purified on a Superdex 30size exclusion column. The pertinent fractions were pooled together,dialyzed against water and frozen until in situ perfusion experiments.

[0074] Both PEG_(2k)-DPDPE and PEG_(5k)-DPDPE were iodinated and testedin in situ perfusion, capillary depletion, brain extraction and proteinbinding studies as in Example 3. A significant increase in brain uptakewas observed for both PEG_(2k)-DPDPE and PEG_(5k)-DPDPE. It wasdetermined that for both of these compounds, the increase in uptake wasdue to peptide entering the brain rather than being trapped in thecapillaries.

EXAMPLE 6 Conjugation of PEG to Biphalin

[0075] a. (mPEG_(2K))₂-Biphalin

[0076] Biphalin (21.1 mg, 0.046 mmol) was dissolved into 15 ml ofanhydrous acetonitrile and treated with 16 μl of triethylamine (0.115mmol, 2.5 fold molar excess). At the same time, mPEG_(2K)-SPA (110 mg,0.055 mmol, 1.2 fold molar excess) was dissolved into 5 ml ofacetonitrile. The dissolved mPEG_(2K)-SPA was slowly added into theabove biphalin solution and the reaction mixture was stirred 66 hours atroom temperature under nitrogen atmosphere.

[0077] Di-pegylated [(mPEG₂K)₂-biphalin] and monopegylated biphalin[mPEG_(2K)-biphalin] were separated from unreacted PEG and free biphalinon a Vydac C18 reverse-phase column at 1 ml/min and 215 nm UV detectorusing a gradient elution of 30% to 60% solvent B. Solvent A is 0.1% TFAin water and solvent B is 0.1% TFA in acetonitrile.

[0078] b. (mPEG_(5K))₂-Biphalin

[0079] Dissolve 118.7 mg mthoxy-PEG_(5K)-SPA (2.374×10⁻⁵ moles, 1.5 foldmolar excess) in 3.0 mL anhydrous acetonitrile. Under a slow Argon flow,add 10.0 mg Biphalin (1.583×10⁻⁵ moles of —NH₂ group) followed bypipette 4.4 L triethylamine (3.166×10⁻⁵ moles, 2.0 fold molar excess)into the solution. Stir at ambient solution for overnight.

[0080] Evaporate solvent on rotary evaporator at 40° C. till neardryness, then further dry on high vacuum for 5 minutes (Use a liquidnitrogen trap when apply vacuum). Dissolve the remaining in 10 mLdeionized water. The solution pH is 4.5. Load the solution by injectioninto a prehydrated Slide-A-Lyzer dialysis cassette with 3500 MWCO (fromPIERCE) and dialysis against 2×900 mL deionized water over three days.

[0081] Load the solution onto 2 mL DEAE Sepharose column. Collect theeluent. Elute the column with additional 125 mL deionized water, andcollect the eluent (pH 7.6). Combine the two fractions, freeze thesolution by liquid nitrogen, and then lyophilize on a freeze dryer.

[0082] c. (mPEG_(12K))₂-Biphalin

[0083] Dissolve 141.4 mg methoxy-PEG_(12K)-SPA (1.187×10⁻⁵ moles, 1.5fold molar excess) in 2.0 mL anhydrous acetonitrile. Under a slow Argonflow, add 5.0 mg Biphalin.2TFA (7.915×10⁻⁶ moles of —NH₂ group) followedby pipette 2.2 μL triethylamine (1.583×10⁻⁵ moles, 2.0 fold molarexcess) into the solution. Stir at ambient solution for overnight.

[0084] Evaporate solvent on high vacuum at room temperature till dryness(Use a liquid nitrogen trap when apply vacuum). Dissolve the remainingin 10 mL deionized water. Load the solution by injection into aprehydrated Dialysis Cassette with 10000 MWCO (from PIERCE) and dialysisagainst 2×800 mL deionized water over three days.

[0085] Dilute the solution to 18 mL by deionized water. Load thesolution onto 10 mL DEAE Sepharose column. Collect the eluent. Elute thecolumn with additional 90 mL deionized water. Combine the fractions,frozen by liquid nitrogen, and then lyophilize on a freeze dryer.

[0086] d. (mPEG_(20K))₂-Biphalin

[0087] Dissolve 255.2 mg Methoxy-PEG_(20K)-SPA (1.187×10⁻⁵ moles, 1.5fold molar excess) in 3.0 mL anhydrous acetonitrile. Under a slow Argonflow, add 5.0 mg Biphalin.2TFA (7.915×10⁻⁶ moles of —NH₂ group) followedby pipette 2.2 μL triethylamine (1.583×10⁻⁵ moles, 2.0 fold molarexcess) into the solution. Stir at ambient solution for overnight.

[0088] Evaporate solvent on high vacuum at room temperature untildryness (Use a liquid nitrogen trap when apply vacuum). Dissolve theremaining in 10 mL deionized water. Load the solution by injection intoa prehydrated Dialysis Cassette with 10000 MWCO (from PIERCE) anddialysis against 2×800 mL deionized water over three days.

[0089] Dilute the solution to 25 mL by deionized water. Load thesolution onto 15 mL DEAE Sepharose column. Collect the eluent. Elute thecolumn with additional 150 mL deionized water. Combine the fractions,frozen by liquid nitrogen, and then lyophilize on a freeze dryer.

[0090] Purity of each sample was determined by reverse-phase HPLC and bymass spectrometry (MALDI).

EXAMPLE 7 Analgesia Assay

[0091] Animals

[0092] Male ICR mice (20-25 g) or male Sprague-Dawley rats (250-300 g)(Harlan Sprague-Dawley Inc., Indianapolis, Ind.) were used for theseexperiments. Animals were housed four per cage in an animal carefacility maintained at 22±0.5° C. with an alternating 12 hr light-darkcycle. Food and water were available ad libitum. Animals were used onlyonce.

[0093] Protocol

[0094] All drugs were dissolved in sterile saline and were prepared sothat the proper dose would be delivered in 5 μl (i.c.v.), 100 μl (i.v.),100 μl (s.c.) and 100 μl (i.m.) of the vehicle. All rodents wererecorded for baseline latency before injection of the drug. A morphinecontrol was used with the i.c.v. and i.v. injection procedures tocompare the analgesic efficacies of test compounds.

[0095] I.C.V. Inection

[0096] Rodents were placed into ajar containing gauze soaked with ethylether until they went into a light sleep. The rodents were immediatelyremoved from the jar and a ½″ incision was made with a scalpel to exposethe top of the skull. The right lateral ventricle was located bymeasuring 2 mm lateral of the midline and 2 mm caudal to Bregma. At thispoint, a Hamilton syringe (22G, ½″) was placed through the skull 2 mmand a 5 μl injection of the compound was delivered. The rodents werethen placed back into their cages until the specified testing time.Methylene blue was placed into the injection site to insure properdelivery of the compound into the lateral ventricle.

[0097] I.V. Injection

[0098] Rodents were placed into a restraint holder and their tails wereplaced into a beaker of warm water and then swabbed with ethanol tomaximize vasodilation in the tail veins. A vein was selected and therestraint was braced to prevent excessive movement. A 30G needle wasselected as the proper size for delivery of the compounds. The needlewas carefully inserted into the vein of each mouse and a 100 μl boluswas slowly delivered. Blanching of the vein up towards the body wasindicative of proper delivery.

[0099] S.C. Injection

[0100] Rats were restrained by hand to prevent excessive movement. A 30Gneedles was selected as the proper size for delivery of the compounds.The needle was carefully inserted into the scruff of the neck of eachrat and a 100 μl bolus was slowly delivered.

[0101] I.M. Injection

[0102] Rats were restrained by hand to prevent excessive movement. A 30Gneedles was selected as the proper size for delivery of the compounds.The needle was carefully inserted into the right hind leg muscle of eachrat and a 100 μl bolus was slowly delivered.

[0103] Analgesia Testing

[0104] The rodents were placed into restraint holders and their tailswere properly placed under the radiant heat beam. The beam was turned onand the time until the animal flicked their tail from under the beam wasrecorded at each time point. In instances where the animals moved theirtails without a flick, the animals were retested only if the elapsedtime under the radiant beam was less than 5 seconds.

[0105] Assessment of Analgesic Data

[0106] The raw data (recorded times) was converted to a percentage ofthe maximum possible effect (% M.P.E.) which was determined as 15seconds. % M.P.E. was determined by the following equation:

% M.P.E.=(Recorded time−Baseline)/(15−Baseline)×100

[0107] These percentages then allow the compound to be plotted accordingto % M.P.E. vs. Time. The curve can then be analyzed to determine thearea under the curve (AUC).

[0108] The results of the i.c.v. administration of the PEG-DPDPE clearlyindicates that PEGylation does not interfere with DPDPE's ability toproduce an analgesic effect. (FIG. 1) Furthermore, the study showed atrend toward a prolongation of analgesic effect of the PEGylatedcompound when compared to the parent compound.

[0109] Intraveneous injection of PEG-DPDPE showed that the PEGylatedcompound is able to cross the blood brain barrier, in sufficientamounts, as to maintain its analgesic properties. (FIG. 2) This studyalso helped confirm that PEGylation for DPDPE significantly prolongs theduration of the analgesic effect.

[0110] All PEGylated biphalin and biphalin samples exhibited a potentanalgesic response in mice with a maximum response of 80-90% reachedbetween 30-45 minutes. The (mPEG_(2K))₂-biphalin continued to prolongthe analgesic effect with a 50% M.P.E. being seen at the 400 minute markof the study as compared to the 90 minute mark for native biphalin. Thedata also shows an inverse relationship between the molecular weight ofPEG and the % M.P.E. (FIG. 3)

[0111] When comparing the analgesic effect of monopegylated biphalin(mPEG_(2K)-biphalin) to that of the dipegylated biphalin[(mPEG_(2K))₂-biphalin] at the same concentration in mice, the durationof analgesic effect for mPEG_(2K)-biphalin is nearly half of that for(mPEG_(2K))₂-biphalin at 50% M.P.E. (FIG. 4) In fact there is nearlyequivalent analgesic effect of mPEG_(2K)-biphalin at half the dose of(mPEG_(2K))₂-biphalin.

[0112] Intravenous administration of (mPEG_(2K))₂-biphalin gives alonger lasting analgesic effect in rats than native biphalin at thevarious doses tested. (FIG. 5) Rats given (mPEG_(2K))₂-biphalin bysubcutaneous or intramuscular administration show elevated and sustainedlevels of analgesic activity as compared to native biphalin at the sameconcentration. (FIG. 6)

EXAMPLE 8 In situ Perfusion Studies of PEG-DPDPE and PEG-Biphalin

[0113] The protocol for the rat brain perfusion experiments was approvedby the Institutional Animal Care and Use Committee at the University ofArizona. The in situ perfusion studies were carried out as previouslyreported (Williams et al., J. Neurochem., 66 (3), pp1289-1299, 1996).PEG_(2K)-DPDPE had a very high in situ uptake R_(Br) value of 3.41±0.15.The in situ perfusion of I¹²⁵ DPDPE is comparable to that of themonopegylated DPDPE, RBr=3.54±0.30. I¹²⁵ labeled Biphalin has an in situperfusion uptake of 7.26±0.11, while the in situ uptake of(mPEG_(2K))₂-Biphalin was dramatically lower, R_(Br) value of 2.70±0.27.

[0114] Many modifications and other embodiments of the invention willcome to mind to one skilled in the art to which this invention pertainshaving the benefit of the teachings presented in the foregoingdescriptions and the associated drawings. Therefore, it is to beunderstood that the invention is not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

What is claimed is:
 1. A substantially hydrophilic conjugate comprising a peptide covalently linked to a water-soluble, nonpeptidic polymer, wherein said peptide is stabilized in circulation and said conjugate can transport across the blood-brain barrier of a mammal.
 2. The conjugate of claim 1 wherein said peptide is an analgesic peptide selected from the group consisting of dynorphins, enkephalins, double enkephalins, endorphins, endomorphins, and analogs and derivatives thereof.
 3. The conjugate of claim 2, wherein said peptide is selected from the group consisting of Met-enkephalin, Leu-enkephalin, endomorphin I, endomorphin II, and analogs or dimeric forms thereof.
 4. The conjugate of claim 2, wherein said peptide is dynorphin A or fragments thereof.
 5. The conjugate of claim 2, wherein said peptide is biphalin.
 6. The conjugate of claim 2, wherein said peptide is DPDPE.
 7. The conjugate of claim 1, wherein said water-soluble, nonpeptidic polymer is polyethylene glycol or a copolymer of polyethylene glycol and polypropylene glycol.
 8. The conjugate of claim 1, wherein said water-soluble, nonpeptidic polymer is polyethylene glycol.
 9. The conjugate of claim 8, wherein said polyethylene glycol is selected from the group consisting of monomethoxypolyethylene glycol, branched polyethylene glycol, polyethylene glycol with degradable linkages in the backbone, homobifunctional polyethylene glycol, heterobifunctional polyethylene glycol, multi-arm polyethylene glycol, pendant polyethylene glycol, and forked polyethylene glycol.
 10. The conjugate of claim 9, wherein said peptide is conjugated to at least one polyethylene glycol molecule.
 11. The conjugate of claim 3, wherein the dimeric form of said peptide has two polyethylene glycol chains covalently attached.
 12. The conjugate of claim 5, wherein said biphalin has two polyethylene glycol chains covalently attached.
 13. The conjugate of claim 8, wherein said polyethylene glycol has a nominal average molecular weight of about 200 daltons to about 100,000 daltons.
 14. The conjugate of claim 13, wherein said polyethylene glycol has a nominal average molecular weight of about 1000 daltons to about 40,000 daltons.
 15. The conjugate of claim 13, wherein said polyethylene glycol has a nominal average molecular weight of 2000 daltons.
 16. A composition comprising a conjugate according to claim 1 and a pharmaceutically acceptable carrier for said conjugate.
 17. A method for delivering a peptide into the brain of an animal through the blood-brain barrier comprising: providing a conjugate between a peptide and a water-soluble polymer, non-peptidic polymer; administering said conjugate into the blood stream of an animal; and transporting the conjugate across the blood-brain barrier of said animal.
 18. The method of claim 18, wherein said peptide is an analgesic peptide.
 19. The method of claim 18, wherein said polymer is selected from the group consisting of copolymers of polyethylene glycol and polypropylene glycol, monomethoxypolyethylene glycol, branched polyethylene glycol, polyethylene glycol with degradable linkages in the backbone, homobifunctional polyethylene glycol, heterobifunctional polyethylene glycol, multi-arm polyethylene glycol, pendant polyethylene glycol, and forked polyethylene glycol.
 20. The method of claim 19, wherein said polyethylene glycol has a nominal average molecular weight from about 200 to about 100,000 daltons.
 21. The method of claim 18, wherein said peptide is an analgesic peptide selected from the group consisting of dynorphins, enkephalins, endorphins, endomorphins, biphalin, and analogs and derivatives thereof.
 22. The method of claim 17, wherein said peptide is conjugated to at least one polymer molecule.
 23. The method of claim 17, wherein said peptide is conjugated to at least two polymer molecules.
 24. The method of claim 17, wherein said step of administering said conjugate comprises parenterally injecting said conjugate into said animal.
 25. The method of claim 17, wherein said step of administering said conjugate comprises of pulmonary and intranasal inhalation into said animal.
 26. The method of claim 17, wherein said step of administering said conjugate is by oral, ocular, buccal, transdermal, or rectal administration.
 27. A method for delivering into the brain of an animal through the blood-brain barrier an agent that is incapable of crossing the blood-brain barrier comprising: providing a conjugate comprising a water-soluble, nonpeptidic polymer, a peptide that is transportable across the blood-brain barrier covalently linked to said polymer, and a nontransportable agent also covalently linked to the polymer; and administering the conjugate into the blood stream of said animal and transporting the conjugate across the blood-brain barrier of said animal.
 28. The method of claim 27, wherein said non-transportable agent is an imaging agent.
 29. The method of claim 27, wherein said non-transportable agent is doxorubicin. 